Immune Escape in Glioblastoma Multiforme and the Adaptation of Immunotherapies for Treatment.

Glioblastoma multiforme (GBM) is the most frequently occurring primary brain tumor and has a very poor prognosis, with only around 5% of patients surviving for a period of 5 years or more after diagnosis. Despite aggressive multimodal therapy, consisting mostly of a combination of surgery, radiotherapy, and temozolomide chemotherapy, tumors nearly always recur close to the site of resection. For the past 15 years, very little progress has been made with regards to improving patient survival. Although immunotherapy represents an attractive therapy modality due to the promising pre-clinical results observed, many of these potential immunotherapeutic approaches fail during clinical trials, and to date no immunotherapeutic treatments for GBM have been approved. As for many other difficult to treat cancers, GBM combines a lack of immunogenicity with few mutations and a highly immunosuppressive tumor microenvironment (TME). Unfortunately, both tumor and immune cells have been shown to contribute towards this immunosuppressive phenotype. In addition, current therapeutics also exacerbate this immunosuppression which might explain the failure of immunotherapy-based clinical trials in the GBM setting. Understanding how these mechanisms interact with one another, as well as how one can increase the anti-tumor immune response by addressing local immunosuppression will lead to better clinical results for immune-based therapeutics. Improving therapeutic delivery across the blood brain barrier also presents a challenge for immunotherapy and future therapies will need to consider this. This review highlights the immunosuppressive mechanisms employed by GBM cancers and examines potential immunotherapeutic treatments that can overcome these significant immunosuppressive hurdles.

Mapping of fine-scale rat prefrontal cortex connections: Evidence for detailed ordering of inputs and outputs connecting the temporal cortex and sensory-motor regions.

Cerebral cortex structure is crucially important for cortical organization and function. The organization of prefrontal cortex (PFC) is controversial and here we seek to understand it more clearly through the study of fine-scale cortical connections. To determine the ordering of microscale input and output connections in the rat PFC, we injected small volumes (20-30 nl) of anterograde (Fluro-Ruby) and retrograde (Fluoro-Gold) neuroanatomical tracers into PFC. These injections revealed several connected regions of the brain but here we report findings restricted to PFC to temporal cortex and sensory-motor cortex pathways. In agreement with previous studies incorporating larger injection volumes we found that smaller injection volumes revealed a more detailed, fine-scale ordering of both prefrontal inputs and output connections to the temporal cortex and sensory-motor cortex regions. These findings are also supported by labelling observed from additional tracer injections made into corresponding regions of temporal cortex. The topography observed reflected the ordering seen at a larger level (i.e., with larger injection volumes) but there were some differences in the topography, such as in relation to the direction of ordering. In agreement with earlier work, we found that fine-scale input and output connections were not always aligned with respect to one another. These results provide evidence for topographically arranged inputs and outputs in two distinct PFC pathways, along with evidence for different connectional patterns within the same pathways. Based on theories of functional connectivity, these findings provide evidence for prefrontal cortical regions residing within networks that contribute to different cognitive functions.

Differences in anatomical connections across distinct areas in the rodent prefrontal cortex.

Prefrontal cortex (PFC) network structure is implicated in a number of complex higher-order functions and with a range of neurological disorders. It is therefore vital to our understanding of PFC function to gain an understanding of its underlying anatomical connectivity. Here, we injected Fluoro-Gold and Fluoro-Ruby into the same sites throughout rat PFC. Tracer injections were applied to two coronal levels within the PFC (anterior +4.7 mm to bregma and posterior +3.7 mm to bregma). Within each coronal level, tracers were deposited at sites separated by approximately 1 mm and located parallel to the medial and orbital surface of the cortex. We found that both Fluoro-Gold and Fluoro-Ruby injections produced prominent labelling in temporal and sensory-motor cortex. Fluoro-Gold produced retrograde labelling and Fluoro-Ruby largely produced anterograde labelling. Analysis of the location of these connections within temporal and sensory-motor cortex revealed a consistent topology (as the sequence of injections was followed mediolaterally along the orbital surface of each coronal level). At the anterior coronal level, injections produced a similar topology to that seen in central PFC in earlier studies from our laboratory (i.e. comparing equivalently located injections employing the same tracer), this was particularly prominent within temporal cortex. However, at the posterior coronal level this pattern of connections differed significantly, revealing higher levels of reciprocity, in both temporal cortex and sensory-motor cortex. Our findings indicate changes in the relative organization of connections arising from posterior in comparison to anterior regions of PFC, which may provide a basis to determine how complex processes are organized.

The topology of connections between rat prefrontal and temporal cortices.

Understanding the structural organization of the prefrontal cortex (PFC) is an important step toward determining its functional organization. Here we investigated the organization of PFC using different neuronal tracers. We injected retrograde (Fluoro-Gold, 100 nl) and anterograde [Biotinylated dextran amine (BDA) or Fluoro-Ruby, 100 nl] tracers into sites within PFC subdivisions (prelimbic, ventral orbital, ventrolateral orbital, dorsolateral orbital) along a coronal axis within PFC. At each injection site one injection was made of the anterograde tracer and one injection was made of the retrograde tracer. The projection locations of retrogradely labeled neurons and anterogradely labeled axon terminals were then analyzed in the temporal cortex: area Te, entorhinal and perirhinal cortex. We found evidence for an ordering of both the anterograde (anterior-posterior, dorsal-ventral, and medial-lateral axes: p < 0.001) and retrograde (anterior-posterior, dorsal-ventral, and medial-lateral axes: p < 0.001) connections of PFC. We observed that anterograde and retrograde labeling in ipsilateral temporal cortex (i.e., PFC inputs and outputs) often occurred reciprocally (i.e., the same brain region, such as area 35d in perirhinal cortex, contained anterograde and retrograde labeling). However, often the same specific columnar temporal cortex regions contained only either labeling of retrograde or anterograde tracer, indicating that PFC inputs and outputs are frequently non-matched.

The topology of connections between rat prefrontal, motor and sensory cortices.

The connections of prefrontal cortex (PFC) were investigated in the rat brain to determine the order and location of input and output connections to motor and somatosensory cortex. Retrograde (100 nl Fluoro-Gold) and anterograde (100 nl Biotinylated Dextran Amines, BDA; Fluorescein and Texas Red) neuronanatomical tracers were injected into the subdivisions of the PFC (prelimbic, ventral orbital, ventrolateral orbital, dorsolateral orbital) and their projections studied. We found clear evidence for organized input projections from the motor and somatosensory cortices to the PFC, with distinct areas of motor and cingulate cortex projecting in an ordered arrangement to the subdivisions of PFC. As injection location of retrograde tracer was moved from medial to lateral in PFC, we observed an ordered arrangement of projections occurring in sensory-motor cortex. There was a significant effect of retrograde injection location on the position of labelled cells occurring in sensory-motor cortex (dorsoventral, anterior-posterior and mediolateral axes p < 0.001). The arrangement of output projections from PFC also displayed a significant ordered projection to sensory-motor cortex (dorsoventral p < 0.001, anterior-posterior p = 0.002 and mediolateral axes p < 0.001). Statistical analysis also showed that the locations of input and output labels vary with respect to one another (in the dorsal-ventral and medial-lateral axes, p < 0.001). Taken together, the findings show that regions of PFC display an ordered arrangement of connections with sensory-motor cortex, with clear laminar organization of input connections. These results also show that input and output connections to PFC are not located in exactly the same sites and reveal a circuit between sensory-motor and PFC.

A role for the CAMKK pathway in visual object recognition memory.

The role of the CAMKK pathway in object recognition memory was investigated. Rats' performance in a preferential object recognition test was examined after local infusion into the perirhinal cortex of the CAMKK inhibitor STO-609. STO-609 infused either before or immediately after acquisition impaired memory tested after a 24 h but not a 20-min delay. Memory was not impaired when STO-609 was infused 20 min after acquisition. The expression of a downstream reaction product of CAMKK was measured by immunohistochemical staining for phospho-CAMKI(Thr177) at 10, 40, 70, and 100 min following the viewing of novel and familiar images of objects. Processing familiar images resulted in more pCAMKI stained neurons in the perirhinal cortex than processing novel images at the 10- and 40-min delays. Prior infusion of STO-609 caused a reduction in pCAMKI stained neurons in response to viewing either novel or familiar images, consistent with its role as an inhibitor of CAMKK. The results establish that the CAMKK pathway within the perirhinal cortex is important for the consolidation of object recognition memory. The activation of pCAMKI after acquisition is earlier than previously reported for pCAMKII.

Differing time dependencies of object recognition memory impairments produced by nicotinic and muscarinic cholinergic antagonism in perirhinal cortex.

The roles of muscarinic and nicotinic cholinergic receptors in perirhinal cortex in object recognition memory were compared. Rats' discrimination of a novel object preference test (NOP) test was measured after either systemic or local infusion into the perirhinal cortex of the nicotinic receptor antagonist methyllycaconitine (MLA), which targets alpha-7 (α7) amongst other nicotinic receptors or the muscarinic receptor antagonists scopolamine, AFDX-384, and pirenzepine. Methyllycaconitine administered systemically or intraperirhinally before acquisition impaired recognition memory tested after a 24-h, but not a 20-min delay. In contrast, all three muscarinic antagonists produced a similar, unusual pattern of impairment with amnesia after a 20-min delay, but remembrance after a 24-h delay. Thus, the amnesic effects of nicotinic and muscarinic antagonism were doubly dissociated across the 20-min and 24-h delays. The same pattern of shorter-term but not longer-term memory impairment was found for scopolamine whether the object preference test was carried out in a square arena or a Y-maze and whether rats of the Dark Agouti or Lister-hooded strains were used. Coinfusion of MLA and either scopolamine or AFDX-384 produced an impairment profile matching that for MLA. Hence, the antagonists did not act additively when coadministered. These findings establish an important role in recognition memory for both nicotinic and muscarinic cholinergic receptors in perirhinal cortex, and provide a challenge to simple ideas about the role of cholinergic processes in recognition memory: The effects of muscarinic and nicotinic antagonism are neither independent nor additive.

Interfering with perirhinal brain-derived neurotrophic factor expression impairs recognition memory in rats.

The role of brain-derived neurotrophic factor (BDNF) in recognition memory was investigated by locally infusing oligodeoxynucleotides (ODNs) into perirhinal cortex, a region of the temporal lobe essential for familiarity discrimination. Antisense but not sense BDNF ODN impaired consolidation of long-term (24 h) but not shorter-term (20 min) recognition memory.

Creating abstract topographic representations: implications for coding, learning and reasoning.

Topographic maps are a fundamental and ubiquitous feature of the sensory and motor regions of the brain. There is less evidence for the existence of conventional topographic maps in associational areas of the brain such as the prefrontal cortex and parietal cortex. The existence of topographically arranged anatomical projections is far more widespread and occurs in associational regions of the brain as well as sensory and motor regions: this points to a more widespread existence of topographically organised maps within associational cortex than currently recognised. Indeed, there is increasing evidence that abstract topographic representations may also occur in these regions. For example, a topographic mnemonic map of visual space has been described in the dorsolateral prefrontal cortex and topographically arranged visuospatial attentional signals have been described in parietal association cortex. This article explores how abstract representations might be extracted from sensory topographic representations and subsequently code abstract information. Finally a simple model is presented that shows how abstract topographic representations could be integrated with other information within the brain to solve problems or form abstract associations. The model uses correlative firing to detect associations between different types of stimuli. It is flexible because it can produce correlations between information represented in a topographic or non-topographic coordinate system. It is proposed that a similar process could be used in high-level cognitive operations such as learning and reasoning.

Coding of distributed, topographic and non-specific representations within the brain.

This article explores the theoretical basis of coding within topographic representations, where neurons encoding specific features such as locations, are arranged into maps. A novel type of representation, termed non-specific, where each neuron does not encode specific features is also postulated. In common with the previously described distributed representations [Rolls, E.T., Treves, A., 1998. Neural Networks and Brain Function. Oxford University Press, Oxford], topographic representations display an exponential relationship between stimuli encoded and both number of neurons and maximum firing rate of those neurons. The non-specific representations described here display a binomial expansion between the number of stimuli encoded and the sum of the number of neurons and the maximum firing rate; therefore groups of non-specific neurons usually encode less stimuli than equivalent topographic layers of neurons. Lower and higher order sensory regions of the brain use either topographic or distributed representations to encode information. It is proposed that non-specific representations may occur in regions of the brain where different types of information may be represented by the same neurons, as occurs in the prefrontal cortex.

Using topographic networks to build a representation of consciousness.

The subject of consciousness has intrigued both psychologists and neuroscientists for many years. Recently, following many recent advances in the emerging field of cognitive neuroscience, there is the possibility that this fundamental process may soon be explained. In particular, there have been dramatic insights gained into the mechanisms of attention, cognition and perception in recent decades. Here, simple network models are proposed which are used to create a representation of consciousness. The models are inspired by the structure of the thalamus and all incorporate topographic layers in their structure. Operation of the models allows filtering of the information reaching the representation according to (1) modality and/or (2) sub-modality, in addition several of the models allowing filtering at the topographic level. The models presented have different structures and employ different integrative mechanisms to produce gating or amplification at different levels; the resultant representations of consciousness are discussed.

Transforming bottom-up topographic representations with top-down signals in the brain.

There has been considerable success in allocating function to the different parts of the brain. We also know much about brain organisation in different regions of the brain and how different brain regions connect to one another. One of the most important next steps for modern neuroscience is to work out how different areas of the brain interact with one another. In particular we need to know how sensory regions communicate with association areas and vice versa. This article explores how top-down signals originating from association areas may be used to process and transform bottom-up representations originating from sensory areas of the brain. Simple models of networks containing topographically organised ensembles of neurons used to integrate and process information are described. The different models can be used to process information in a variety of different ways that could be used as the starting point for a variety of cognitive operations, in particular the extraction of abstract information from sensory representations.

Selective representation of task-relevant objects and locations in the monkey prefrontal cortex.

In the monkey prefrontal cortex (PFC), task context exerts a strong influence on neural activity. We examined different aspects of task context in a temporal search task. On each trial, the monkey (Macaca mulatta) watched a stream of pictures presented to left or right of fixation. The task was to hold fixation until seeing a particular target, and then to make an immediate saccade to it. Sometimes (unilateral task), the attended pictures appeared alone, with a cue at trial onset indicating whether they would be presented to left or right. Sometimes (bilateral task), the attended picture stream (cued side) was accompanied by an irrelevant stream on the opposite side. In two macaques, we recorded responses from a total of 161 cells in the lateral PFC. Many cells (75/161) showed visual responses. Object-selective responses were strongly shaped by task relevance - with stronger responses to targets than to nontargets, failure to discriminate one nontarget from another, and filtering out of information from an irrelevant stimulus stream. Location selectivity occurred rather independently of object selectivity, and independently in visual responses and delay periods between one stimulus and the next. On error trials, PFC activity followed the correct rules of the task, rather than the incorrect overt behaviour. Together, these results suggest a highly programmable system, with responses strongly determined by the rules and requirements of the task performed.

Gain control from beyond the classical receptive field in primate primary visual cortex.

Gain control is a salient feature of information processing throughout the visual system. Heeger (1991, 1992) described a mechanism that could underpin gain control in primary visual cortex (V1). According to this model, a neuron's response is normalized by dividing its output by the sum of a population of neurons, which are selective for orientations covering a broad range. Gain control in this scheme is manifested as a change in the semisaturation constant (contrast gain) of a V1 neuron. Here we examine how flanking and annular gratings of the same or orthogonal orientation to that preferred by a neuron presented beyond the receptive field modulate gain in V1 neurons in anesthetized marmosets (Callithrix jacchus). To characterize how gain was modulated by surround stimuli, the Michaelis-Menten equation was fitted to response versus contrast functions obtained under each stimulus condition. The modulation of gain by surround stimuli was modelled best as a divisive reduction in response gain. Response gain varied with the orientation of surround stimuli, but was reduced most when the orientation of a large annular grating beyond the classical receptive field matched the preferred orientation of neurons. The strength of surround suppression did not vary significantly with retinal eccentricity or laminar distribution. In the marmoset, as in macaques (Angelucci et al., 2002a,b), gain control over the sort of distances reported here (up to 10 deg) may be mediated by feedback from extrastriate areas.

The nature of V1 neural responses to 2D moving patterns depends on receptive-field structure in the marmoset monkey.

A plaid pattern is formed when two sinusoidal gratings of different orientations are added together. Previous work has shown that V1 neurons selectively encode the direction and orientation of the component gratings in a moving plaid but not the direction of the plaid itself (Movshon et al. 1985). We recorded the responses of 49 direction-selective neurons to moving gratings and plaid patterns in area V1 of the anesthetized marmoset monkey (Callithrix jacchus). The responses of V1 neurons to rectangular patches of varying lengths and widths containing gratings of optimal spatial frequency were used to measure size and aspect ratio of the receptive-field subunits. We measured responses to plaid patterns moving in different directions and graded the magnitude of the response to the direction of motion of the plaid and the response to the direction of motion of the component gratings. We found significant correlations between receptive-field structure and the type and strength of its response to moving plaid patterns. The strength of pattern and component responses was significantly correlated with the interrelated properties of direction tuning width (Spearman's r = 0.82, P < 0.001), and receptive-field subunit aspect ratio (Spearman's r = -0.79, P < 0.001). Neurons with broad direction tuning and short, wide receptive-field subunits gave their greatest response when the plaid moved in their preferred direction. Conversely, neurons with narrow direction tuning and long, narrow receptive-field subunits gave their greatest responses when the plaid moved in a direction such that one of its components moved in the preferred direction.

The uses of colour vision: behavioural and physiological distinctiveness of colour stimuli.

Colour and greyscale (black and white) pictures look different to us, but it is not clear whether the difference in appearance is a consequence of the way our visual system uses colour signals or a by-product of our experience. In principle, colour images are qualitatively different from greyscale images because they make it possible to use different processing strategies. Colour signals provide important cues for segmenting the image into areas that represent different objects and for linking together areas that represent the same object. If this property of colour signals is exploited in visual processing we would expect colour stimuli to look different, as a class, from greyscale stimuli. We would also expect that adding colour signals to greyscale signals should change the way that those signals are processed. We have investigated these questions in behavioural and in physiological experiments. We find that male marmosets (all of which are dichromats) rapidly learn to distinguish between colour and greyscale copies of the same images. The discrimination transfers to new image pairs, to new colours and to image pairs in which the colour and greyscale images are spatially different. We find that, in a proportion of neurons recorded in the marmoset visual cortex, colour-shifts in opposite directions produce similar enhancements of the response to a luminance stimulus. We conclude that colour is, both behaviourally and physiologically, a distinctive property of images.

Filtering of neural signals by focused attention in the monkey prefrontal cortex.

Prefrontal cortex is thought to be important in attention and awareness. Here we recorded the activity of prefrontal neurons in monkeys carrying out a focused attention task. Having directed attention to one location, monkeys monitored a stream of visual objects, awaiting a predefined target. Although neurons rarely discriminated between one non-target and another, they commonly discriminated between targets and non-targets. From the onset of the visual response, this target/non-target discrimination was effectively eliminated when the same objects appeared at an unattended location in the opposite visual hemifield. The results show that, in prefrontal cortex, filtering of ignored locations is strong, early and spatially global. Such filtering may be important in blindness to unattended signals--a conspicuous aspect of human selective attention.

Feedback from V1 and inhibition from beyond the classical receptive field modulates the responses of neurons in the primate lateral geniculate nucleus.

It is well established that the responses of neurons in the lateral geniculate nucleus (LGN) can be modulated by feedback from visual cortex, but it is still unclear how cortico-geniculate afferents regulate the flow of visual information to the cortex in the primate. Here we report the effects, on the gain of LGN neurons, of differentially stimulating the extraclassical receptive field, with feedback from the striate cortex intact or inactivated in the marmoset monkey, Callithrix jacchus. A horizontally oriented grating of optimal size, spatial frequency, and temporal frequency was presented to the classical receptive field. The grating varied in contrast (range: 0-1) from trial to trial, and was presented alone, or surrounded by a grating of the same or orthogonal orientation, contained within either a larger annular field, or flanks oriented either horizontally or vertically. V1 was ablated to inactivate cortico-geniculate feedback. The maximum firing rate of LGN neurons was greater with V1 intact, but was reduced by visually stimulating beyond the classical receptive field. Large horizontal or vertical annular gratings were most effective in reducing the maximum firing rate of LGN neurons. Magnocellular neurons were most susceptible to this inhibition from beyond the classical receptive field. Extraclassical inhibition was less effective with V1 ablated. We conclude that inhibition from beyond the classical receptive field reduces the excitatory influence of V1 in the LGN. The net balance between cortico-geniculate excitation and inhibition from beyond the classical receptive field is one mechanism by which signals relayed from the retina to V1 are controlled.